The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas.

We analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas and found a marked induction of insulin receptor substrate-2 (IRS2) and phosphorylated AKT and a concomitant upregulation of downstream effectors in most leiomyosarcomas. To determine the role of aberrant PI3K-AKT signaling in leiomyosarcoma pathogenesis, we genetically inactivated Pten in the smooth muscle cell lineage by cross-breeding Pten(loxP/loxP) mice with Tagln-cre mice. Mice carrying homozygous deletion of Pten alleles developed widespread smooth muscle cell hyperplasia and abdominal leiomyosarcomas, with a very rapid onset and elevated incidence (approximately 80%) compared to other animal models. Constitutive mTOR activation was restricted to the leiomyosarcomas, revealing the requirement for additional molecular events besides Pten loss. The rapamycin derivative everolimus substantially decelerated tumor growth on Tagln-cre/Pten(loxP/loxP) mice and prolonged their lifespan. Our data show a new and critical role for the AKT-mTOR pathway in smooth muscle transformation and leiomyosarcoma genesis, and support treatment of selected sarcomas by the targeting of this pathway with new compounds or combinations of these with conventional chemotherapy agents.

TMPRSS2-ERG rearrangement in dominant anterior prostatic tumours: incidence and correlation with ERG immunohistochemistry.

AIM: To study prostate cancer zonal differences in TMPRSS2-ERG gene rearrangement. METHODS AND RESULTS: We examined 136 well-characterized dominant anterior prostatic tumours, including 61 transition zone (TZ) and 75 anterior peripheral zone (PZ) lesions, defined using strict anatomical considerations. TMPRSS2-ERG FISH and ERG protein immunohistochemistry were performed on tissue microarrays. FISH results, available for 56 TZ and 71 anterior PZ samples, were correlated with ERG staining and TZ-associated 'clear cell' histology. Fewer TZ cancers (four of 56; 7%) were rearranged than anterior PZ cancers (18 of 71; 25%) (P = 0.009); deletion was the sole mechanism of TZ cancer rearrangement. ERG protein overexpression was present in 4% (two of 56; both FISH+) and 30% (21 of 71; 17 FISH+) of TZ and anterior PZ tumours, respectively. 'Clear cell' histology was present in 21 of 56 (38%) TZ and eight of 71 (11%) anterior PZ tumours. Seven per cent of cancers with and 21% without this histology had rearrangement, regardless of zonal origin. CONCLUSIONS: TMPRSS2-ERG rearrangement occurs in dominant TZ and anterior PZ prostate cancers, with all rearranged TZ cancers in this cohort showing deletion. ERG immunohistochemistry demonstrated excellent sensitivity (86%) and specificity (96%) for TMPRSS2-ERG rearrangement. TMPRSS2-ERG fusion is rare in TZ tumours and present at a low frequency in tumours displaying 'clear cell' histology.

Clear-cell papillary renal cell carcinoma: molecular and immunohistochemical analysis with emphasis on the von Hippel-Lindau gene and hypoxia-inducible factor pathway-related proteins.

Over the past few years several investigators have independently described unique low-grade renal epithelial neoplasms with clear cytoplasm, focal to diffuse papillary architecture, and occasional leiomyomatous stromal metaplasia that are not currently recognized in the World Health Organization classification of renal tumors. These tumors have been referred to by a variety of names including clear-cell papillary renal cell carcinoma and recently "clear-cell tubulopapillary renal cell carcinoma". On the basis of the available data, such tumors are positive for cytokeratin 7 (CK7) and carbonic anhydrase IX (CA9), while being negative for CD10, α-methylacyl-CoA racemase (AMACR), and TFE3. These tumors reportedly lack trisomies of chromosomes 7 and 17, deletions of 3p25, von Hippel-Lindau (VHL) gene mutations, and VHL promoter hypermethylation. Herein, we report on nine cases of this tumor emphasizing detailed studies of the VHL gene and hypoxia-inducible factor (HIF) pathway. Molecular studies performed included VHL mutational analysis, copy number changes assessed using single-nucleotide polymorphism arrays, and qRT-PCR for VHL mRNA expression. Immunohistochemical stains for markers of HIF pathway activation (HIF-1α, CA9, and glucose transporter-1 (GLUT-1)) as well as other relevant markers (CK7, CD10, AMACR, and TFE3) were performed. None of our tumors harbored VHL gene mutations, losses of chromosomal region 3p25, or trisomies of chromosomes 7 or 17. VHL mRNA was overexpressed in our tumors relative to normal renal tissue and clear-cell renal cell carcinoma. All cases showed strong co-expression of CK7, HIF-1α, GLUT-1, and CA9. No expression of TFE3, CD10, or AMACR was seen. The morphological, immunophenotypic, and molecular features of these unique low-grade tumors are sufficiently distinct to allow separation from other renal cell carcinoma subtypes. The co-expression of CA9, HIF-1α, and GLUT-1 in the absence of VHL gene alterations in clear-cell papillary renal cell carcinoma suggests activation of the HIF pathway by non-VHL-dependent mechanisms.

Hypoxia-inducible factor and mammalian target of rapamycin pathway markers in urothelial carcinoma of the bladder: possible therapeutic implications.

OBJECTIVE: To investigate the rationale for using targeted therapies against hypoxia-inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways in urothelial carcinoma of the bladder, by studying the immunohistochemical expression of molecules of these pathways in urothelial carcinoma, as recent pre-clinical studies and clinical trials have shown the potential utility of such targeted therapies. PATIENTS AND METHODS: Immunohistochemical stains were performed on a tissue microarray prepared from 92 cases of ≥ pT2 urothelial (transitional cell) carcinoma of bladder, using antibodies against HIF-1α and VEGF-R2, and phospho-S6 and phospho-4E BP1, molecules of HIF and activated mTOR pathways, respectively. Immunoreactivity was graded from 0 to 3+ (0, 0-5%; 1+, 6-25%; 2+, 26-50%; 3+, > 50% tumour cells positive). RESULTS: In all, 58, 34, 35 and 17% of the tumours showed grade 2-3+ expression of phospho-4E BP1, phospho-S6, HIF-1α and VEGF-R2, respectively. Moderate correlation for immunoreactivity was observed between molecules within the same pathway [(phospho-4E BP1 with phospho-S6 (rho = 0.411), and HIF-1α with VEGF-R2 (rho = 0.265)], but not between molecules across pathways. CONCLUSIONS: Urothelial carcinomas of the bladder express molecules of the HIF and mTOR pathways, providing a rationale for clinical trials evaluating agents targeting these pathways. Correlation between molecules within the same pathway, and not across pathways, suggests that investigating the usefulness of a specific targeted agent might benefit from pre-treatment evaluation of pathway marker expression.

Parathyroid neoplasms: clinical, histopathological, and tissue microarray-based molecular analysis.

We studied 45 patients with typical and 8 with atypical parathyroid adenomas as well as 20 with parathyroid carcinomas. Clinical, pathological, and molecular analyses were conducted on all adenomas. Clinical data were analyzed for 20, histopathologic slides for 16, and tissue specimens for 8 patients with carcinoma. Molecular expression profiles were investigated by immunohistochemistry (IHC) for Ki-67, p53, mdm2, p21, Bcl-2, cyclin D1, and p27 on paraffin-embedded tissues arrayed on tissue microarrays. Trabecular growth and vascular, capsular, and soft-tissue invasion were characteristic of parathyroid carcinomas but not of typical adenomas. No adenomas recurred. Seventy-four percent of carcinomas recurred, most in the neck. Seventy-nine percent of patients with such illness died of disease after an indolent, multiply recurrent course responsive to repeated resections; the 5-year survival rate was 50%. High Ki-67 proliferative index was seen in 2% of adenomas and 25% of carcinomas, whereas p27 expression was present in 80% of adenomas and 18% of carcinomas. The molecular phenotype, p27(+)Bcl-2(+)Ki-67(-)mdm2(+), was observed in 76%, 29%, and 0% of typical and atypical adenomas and carcinomas, respectively. The complexity of molecular phenotypes increased with tumor aggressiveness. Parathyroid carcinoma is an aggressive disease with a propensity for multiple recurrences. It is characterized by capsular, vascular, and soft-tissue invasion. Recurrence portends poor outcome. Molecular markers, Ki-67 and p27, may distinguish parathyroid carcinoma from adenoma. The molecular phenotype, p27(+)Bcl-2(+)Ki-67(-)mdm2(+), appears to be unique to nonmalignant parathyroid tumors, and multimarker phenotypes are more complex in carcinomas.

Squamous cell carcinoma-related oncogene is highly expressed in developing, normal, and adenomatous adrenal tissue but not in aggressive adrenocortical carcinomas.

BACKGROUND: Our previous work has demonstrated squamous cell carcinoma-related oncogene (SCCRO) expression in adult murine adrenocortical tissue. The aim of this study was to assess patterns of SCCRO expression in the embryonic murine adrenal gland, and in normal and neoplastic human adrenocortical tissues in order to determine its role as a marker of differentiation in adrenocortical development and neoplastic progression. METHODS: Murine embryos were procured at developmental stages E8 to E18. A tissue microarray was constructed containing 38 normal, 39 adenomatous, and 87 carcinomatous human adrenocortical specimens. Immunohistochemistry for SCCRO was performed and its expression was graded in suitable tissues. RESULTS: SCCRO expression was detected in the murine adrenal cortex as early as E15 and persisted into the postnatal period. High-level SCCRO expression was identified in 94% of normal (32/34) and adenomatous (29/31) adrenocortical specimens but in only 63% (45/72) of adrenocortical carcinoma (ACC) specimens ( P = .001). Loss of SCCRO expression in primary ACC (13/34 (34%)) correlated with advanced stage ( P = .06), presence of M1 disease at presentation ( P = .03), and worse overall survival ( P = .006). CONCLUSIONS: SCCRO appears to be a marker of adrenocortical differentiation in both murine and human systems. SCCRO expression may be useful in distinguishing adrenocortical adenomas from ACC. Moreover, loss of SCCRO expression in primary ACC is associated with worse outcome and may be a marker of progressive dedifferentiation in these tumors.

Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cancer: recognition of the syndrome by pathologic features and the utility of detecting aberrant succination by immunohistochemistry.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome is an autosomal dominant disorder in which germline mutations of fumarate hydratase (FH) gene confer an increased risk of cutaneous and uterine leiomyomas and renal cancer. HLRCC-associated renal cancer is highly aggressive and frequently presents as a solitary mass. We reviewed the clinicopathologic features of 9 patients with renal tumors presenting as sporadic cases but who were later proven to have FH germline mutations. Histologically, all tumors showed mixed architectural patterns, with papillary as the dominant pattern in only 3 cases. Besides papillary, tubular, tubulopapillary, solid, and cystic elements, 6 of 9 tumors contained collecting duct carcinoma-like areas with infiltrating tubules, nests, or individual cells surrounded by desmoplastic stroma. Prominent tubulocystic carcinoma-like component and sarcomatoid differentiation were identified. Although all tumors exhibited the proposed hallmark of HLRCC (large eosinophilic nucleolus surrounded by a clear halo), this feature was often not uniformly present throughout the tumor. Prior studies have shown that a high level of fumarate accumulated in HLRCC tumor cells causes aberrant succination of cellular proteins by forming a stable chemical modification, S-(2-succino)-cysteine (2SC), which can be detected by immunohistochemistry. We thus explored the utility of detecting 2SC by immunohistochemistry in the differential diagnosis of HLRCC tumors and other high-grade renal tumors and investigated the correlation between 2SC staining and FH molecular alterations. All confirmed HLRCC tumors demonstrated diffuse and strong nuclear and cytoplasmic 2SC staining, whereas all clear cell (184/184, 100%), most high-grade unclassified (93/97, 96%), and the large majority of "type 2" papillary (35/45, 78%) renal cell carcinoma cases showed no 2SC immunoreactivity. A subset of papillary (22%) and rare unclassified (4%) tumors showed patchy or diffuse cytoplasmic staining without nuclear labeling, unlike the pattern seen with confirmed HLRCC tumors. Sequencing revealed no germline or somatic FH alterations in 14 tumors that either exhibited only cytoplasmic 2SC staining (n=5) or were negative for 2SC (n=9), despite their HLRCC-like morphologic features. Our results emphasize the pivotal role of pathologic examination in the diagnosis of HLRCC patients and indicate immunohistochemical detection of 2SC as a useful ancillary tool in the differentiation of HLRCC renal tumors from other high-grade renal cell carcinomas.

Adrenocortical adenoma and carcinoma: histopathological and molecular comparative analysis.

We compared histomorphological features and molecular expression profiles of adrenocortical adenomas (ACAd) and carcinomas (ACCa). A critical histopathological review (mean, 11 slides per patient) was conducted of 37 ACAd and 67 ACCa. Paraffin-embedded tissue cores of ACAd (n = 33) and ACCa (n = 38) were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, p27, and Ki-67 were investigated by immunohistochemistry and correlated with histopathology and patient outcome using standard statistical methodology. Median follow-up period was 5 years. Tumor necrosis, atypical mitoses, and >1 mitosis per 50 high-power fields were factors that were highly specific for ACCa (P <.001). Number (0 to 4) of unfavorable markers [Ki-67 (+), p21 (+), p27 (+), mdm-2(-)] expressed was significantly associated with mitotic activity and morphologic index (i.e., number of adverse morphologic features) and highly predictive of malignancy (P <.001). Ki-67 overexpression occurred in 0 ACAd and 36% ACCa (P <.001) and was significantly associated with mitotic rate and unfavorable morphologic index (P <.001). Tumor necrosis, atypical mitoses, >5 mitoses per 50 high-power fields, sinusoidal invasion, histologic index of >5, and presence of more than two unfavorable molecular markers were associated significantly with metastasis in ACCa. Well-established histopathologic criteria and Ki-67 can specifically distinguish ACCAd from ACCa. Tumor cell proliferation (Ki-67) correlates with mitotic activity and morphologic index. Tumor morphology is a better predictor of metastatic risk in ACCa than current immunohistochemistry-detected cell cycle regulatory and proliferation-associated proteins.

Clinical significance of molecular expression profiles of Hürthle cell tumors of the thyroid gland analyzed via tissue microarrays.

Hürthle cell tumors are rare thyroid neoplasms for which disease biology is poorly understood and diagnosis of carcinoma can be challenging. The aim of the study was to characterize molecular expression profiles of Hürthle cell tumors and to determine the clinical significance of identified phenotypes. Paraffin-embedded tissue cores of normal thyroid (n = 18), and histopathologically well-defined Hürthle cell adenomas (n = 27), Hürthle cell tumors of unknown malignant behavior (n = 7), and minimally (n = 14) and widely (n = 21) invasive Hürthle cell carcinomas were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, and Ki-67 were detected by immunohistochemistry and correlated with clinicopathological data and patient outcome using standard statistical methodology. Median follow-up time was 8 years. High Ki-67 proliferative index was evident only in the clinically aggressive widely invasive Hürthle cell carcinomas and was associated with significantly reduced relapse-free (P = 0.001) and disease-specific (P < 0.001) survival. The molecular phenotype of Hürthle cell tumors, independent of histopathological subtype diagnosis, was characterized by p53(-), mdm-2(+), p21(+/-), cyclin D1(-), and Bcl-2(+/-). Normal thyroid tissue demonstrated a p53(-), mdm-2(-), p21(-), cyclin D1(-), and Bcl-2(+) phenotype. The Bcl-2(+) phenotype was associated with improved relapse-free survival (P = 0.04) and disease-specific survival (P = 0.01) in widely invasive carcinomas and the Ki-67(+)/Bcl-2(-) phenotype was associated with the diagnosis of widely invasive Hürthle cell carcinoma (P < 0.001). This study demonstrates that tissue microarray-based profiling allows identification of molecular markers that are associated with patient prognosis. High Ki-67 proliferative index was associated with adverse outcome in Hürthle cell neoplasms. Together with down-regulation of Bcl-2, high Ki-67 proliferative index may be useful for diagnosing widely invasive Hürthle cell carcinomas. Molecular alterations in the p53 pathway play a role in Hürthle cell tumorigenesis, but other unidentified molecular changes seem to be required to induce the malignant phenotype.

SCCRO expression correlates with invasive progression in bronchioloalveolar carcinoma.

BACKGROUND: Overexpression of squamous cell carcinoma-related oncogene (SCCRO) is associated with invasive progression and poor outcomes in non-small cell lung cancer. We assessed the role of SCCRO as a tumor marker in bronchioloalveolar carcinoma (BAC), a subtype of adenocarcinoma exhibiting evidence of histologic tumor progression. We hypothesized that SCCRO expression would correlate with invasive tumor phenotypes and worse survival in BAC. METHODS: We classified 150 tumors as pure BAC, BAC with focal invasion, or adenocarcinoma with BAC features. A tissue microarray was constructed from areas of benign lung, BAC, and invasive adenocarcinoma in these tumors. Squamous cell carcinoma-related oncogene expression was graded by immunohistochemistry from 0 to 3 (absent, low, moderate, or high), with positive SCCRO phenotype defined as grade 3. Squamous cell carcinoma-related oncogene specificity was determined by Wilcoxon rank test and area under the receiver-operator curve, survival by the Kaplan-Meier method, and correlation with prognostic factors by log-rank test. RESULTS: Of the 86.0% (129 of 150) of specimens suitable for analysis, positive SCCRO phenotype was seen in 16.3% (21 of 129) and was 100.0% specific for tumor versus benign tissue (area under receiver-operator curve, 0.92). Positive SCCRO phenotype was greater in tumors with increasing degrees of invasive histologic type (7.0% pure BAC, 13.6% BAC with focal invasion, and 28.6% adenocarcinoma with BAC features; p = 0.02). Low-level SCCRO expression was present in 83.9% (99 of 118) of benign tissues and correlated with tobacco use and poor survival (p = 0.05). CONCLUSIONS: Squamous cell carcinoma-related oncogene is a marker of invasive tumor progression in BAC. Low-level expression in adjacent benign lung predicts worse survival, and may represent field cancerization or host-tumor effects.